Method for preparation of highly pure asiaticoside composition from centella asiatica and a method of use thereof

ABSTRACT

Disclosed is a commercially viable asiaticoside composition having at least 99% purity that is derived from plant  Centella asiatica  and a manufacturing process thereof. Also disclosed is a method and the models for oral administration of therapeutically effective amounts of the asiaticoside composition for treatment of Inflammatory Bowel Diseases such as Ulcerative colitis, Crohn&#39;s disease and associated complications of inflammatory bowel diseases such as hemorrhoids, anal fissures, fistulas. Also disclosed is a method and model for oral administration of therapeutically effective amounts of the asiaticoside composition for treatment of  Helicobacter Pylori , as well as a method and the models for oral administration of therapeutically effective amounts of the asiaticoside composition for prevention of colon cancer, gastric diseases and gastric carcinoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/702,834, filed Dec. 7, 2012, which is a United States nationalphase application under 35 U.S.C. §371 of international Application No.PCT/IN2010/000575 filed Aug. 31, 2010, and claims priority under 35U.S.C. §119(a)-(d) to Indian Patent Application No. 1760/MUM/2010, filedJun. 10, 2010, the disclosures of which are each incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an extract of asiaticoside from CentellaAsiatica, and more particularly, use thereof for management ofinflammatory bowel diseases, treatment of Helicobacter Pylori,prevention of gastric diseases and gastric carcinoma.

2. Description of Related Art

Asiaticoside extracted from Centella asiatica is a commerciallyavailable analytical reference standard material with companies such as,for example, Yick-Vic Company having attributed Asiaticoaside purity of92.8% by HPLC, Tauto Biotech Company having attributed Asiaticoasidepurity around 97% by HPLC, and Baoji herbest Biotech Company havingAsiaticoaside assay more than 98%. However, these products arespecifically adapted to be used as a reference standard for researchpurposes in the research laboratories and not found to be an extract ofplant centella asiatica. These asiaticoside products are notcommercially viable to be used as a pharmaceutical composition.

The processes adapted for preparation of asiaticoside from centellaasiatica claim purity ranging between 50%-97% in the prior art. Forexample, US Publication No. 2008/0194499 teaches pharmaceuticalpentacyclic terpenoid glycosides that include asiaticoside compositionranging between 15-50%. Also, US Publication No. 2006/0177516 disclosesa terpenic mixture having asiaticoside composition around 40%. Moreover,U.S. Pat. No. 6,417,349 provides a centella asiatica extract havingrange 4:6 to 6:4 to constitute 97% or more of the extract. In addition,Chinese Patent No. CN 1520824 discloses an extraction and separationmethod for asiaticoside that has purity 92.8%. However, none of theprior art has attempted exclusive extraction of highly pure asiaticosidefrom centella asiatica.

The traditional use of Centella Asiatica or Asiaticoside includespromoting wound healing, treatment of skin diseases, skin disorders andchronic inflammatory diseases. For example, Chinese patent no. CN101129393 discloses the use of asiaticoside liquid in wound healing. Inaddition, US Publication No. 2009/0060985 teaches uses of centellaasiatica urban extract as a drug substance for treatment of skindisorders. There are few attempts seen in the art wherein theasiaticoside or centella asiatica is employed for non-traditional uses.For example, US Publication No. 2008/0194499 teaches the use ofterpenoid glycosides, preferably asiaticoside and madecassosideoptionally along with excipients, for management of depression. Also, USPublication No. 2006/0177516 provides a food supplement that shows usesof Asiaticoside for treatment of anemia conditions.

However, the use of asiaticoside or centella asiatica for management ofdiseases related to gastrointestinal tract such as inflammatory boweldisease and helicobacter pylori is relatively unknown. There are fewattempts seen in the art wherein the asiaticoside is used for treatmentof liver diseases. For example, Chinese patent nos. CN1439376 and CN1520824 disclose the use of asiaticoside for preventing and treatingfibrosis of liver. In addition, US Publication No. 2004/0097463discloses the use of asiaticoside for treatment of cancer associatedwith liver, colon and pancreas. In addition, Cheng et al., 2004, (“Thehealing effects of Centella extract and asiaticoside on acetic acidinduced gastric ulcers in rats”, Life Sciences, vol. 74, pp. 2237-2249)discusses the healing effects of Centella asiatica water extract onacetic acid induced gastric ulcers in rats. Also, Cheng et al., 2000,(“Effects of Centella asiatica on ethanol induced gastric mucosallesions in rats”, Life Sciences, vol. 67, pp. 2647-2653) teachespreventive effect of water extract of Centella asiatica on ex-vivoexperiments of ethanol induced gastric mucosal lesions. However, thegastric ulcers stated in the art are restricted to chemical or druginduced ulceration models. Moreover, these models specifically create alesion by local application of an irritant (acetic acid) in the stomachwhich may not necessarily address the diseases of the colon such asinflammatory bowel diseases. Moreover, the asiaticoside products in theart claim for treatment of ulcers, but fail to specify the use ofasiaticoside for prevention of ulcers.

Inflammatory Bowel Disease is characterized by intractable, chronicinflammatory conditions such as Ulcerative Colitis and Crohn's Diseasewhich display distressing symptoms of abdominal pain, diarrhea,vomiting, hematochezia (blood in stools), reduced appetite, weight loss,fever and various associated complications such as anal fissures,fistulas, perirectal abscess, hemorrhoids, for example. UlcerativeColitis and Crohn's Disease are usually assessed by disease activityindex, which includes stool frequency, presence of blood in stool,endoscopic appearance, and physician's global assessment. Persistence ofabove conditions leads to chronic inflammation and subsequently becomesthe causative factors for development of colonic cancers. The existingmethods of treatment for inflammatory bowel diseases include thereduction of abdominal pain, diarrhea, fatigue, anemia, nutrientdeficiencies, mucosal inflammation, extra intestinal manifestations,hospitalizations, operations, and complications, such as abscesses,fistulae, infections, and malignancy.

The treatment of inflammatory bowel diseases consists of oraladministration of sulfasalazine, immunosupressants and corticosteroids.Normally, Sulfasalazine is a preferred first line treatment for mild tomoderate Ulcerative Colitis and Crohn's Disease. However, the sideeffects such as drug intolerance, impaired folic acid absorption, renaladverse effects make Sulfasalazine undesirable for inflammatory boweldisease treatment. Further, the availability of Sulfasalazine at thecolon is limited due to its absorption in the stomach and subsequentexcretion in the urine. Ulcerative Colitis and Crohn's Disease, in acuteconditions, require treatment with corticosteroids but these drugscannot prevent remission. Long term use of corticosteroids is associatedwith skin thinning, susceptibility to laceration, weight gain, increasein blood pressure, diabetes and related adverse conditions.Immunosuppressants are effective in long term treatment of UlcerativeColitis and Crohn's Disease. These drugs also have significant adversereactions such as bone marrow suppression, lymphomas (in renaltransplant patients), skin cancer and pancreatitis. The inflammatorybowel disease require surgery in severe cases, such as bowel resection,stricture plasty or a temporary or permanent colostomy or ileostomywhich involves use of general anesthesia and complications of postoperative recovery. Ulceration of gastrointestinal tract by mucosaldamage is an associated complication of inflammatory bowel disease. Thechronic nature of inflammatory bowel disease and associated inflammatorydiseases require long term management therapy. Current methods oftreatment of inflammatory bowel disease have limitations of adverseside-effects on sustained treatment and high risk of remission of thedisease.

The inflammations that are induced by pathogenic bacteria such ashelicobacter pylori help facilitate to aggravate the symptoms ofinflammatory bowel diseases. Moreover, the environmental factors such asstress, food and alcohol consumption amplify the activity ofhelicobacter pylori. The World Health Organization (WHO) has categorizedhelicobacter pylori as group-I carcinogen for promoting gastriccarcinoma. The helicobacter pylori infection triggers chronicinflammatory reaction that damages epithelial cell followed by inductionof gastric atrophy that eventually leads to gastric carcinoma. Thetreatment of gastric carcinoma or gastric cancer has highly fataltreatment options such as, for example, surgical removal of canceroustissue by gastrectomy, removal of part/total stomach, chemotherapy,radiation and chemo radiation. However, these treatments havesubstantially high side-effects that lead poor quality of life to thepatients. Also, these treatments do no assure complete recovery as thereis a high probability of recurrence of the carcinoma. Timely diagnosisand treatment for eradication of helicobacter pylori from infectedgastric mucosa may greatly reduce the risk of gastric carcinoma. A usermay appreciate an effective means for eradication of helicobacter pyloriinfection, which can indirectly help in prevention of gastric carcinomainduced by helicobacter pylori infection.

A commercially viable method for extraction of asiaticoside fromcentella asiatica is needed that has a very high purity to beeffectively used as a pharmaceutical composition. An asiaticosidecomposition is further needed that provides a kinder and gentler methodfor effective long-term management of inflammatory bowel disease alongwith helicobacter pylori. An asiaticoside composition is further neededthat prevents gastric diseases and colon cancers that are induced due toinflammatory bowel diseases. An asiaticoside composition is furtherneeded for effective eradication of the helicobacter pylori infectionthat prevents a gastric carcinoma.

SUMMARY OF THE INVENTION

The main object of the present invention is to provide a method orprocess of preparation of a pharmaceutical composition of asiaticosidederived from Centella Asiatica that has at least 99% purity.

Another object of the present invention is to provide the pharmaceuticalcomposition of asiaticoside, optionally along with acceptablepharmaceutical excipients, for treatment of Inflammatory Bowel Diseasessuch as Ulcerative Colitis, Crohn's Disease.

Yet another object of the present invention is to provide thepharmaceutical composition of asiaticoside, optionally along withacceptable pharmaceutical excipients, for treatment of associatedcomplications of inflammatory bowel diseases such as but not limited tohemorrhoids, anal fissures, fistulas.

Still another object of the present invention is to provide thepharmaceutical composition of asiaticoside, optionally along withacceptable pharmaceutical excipients, for prevention of colon cancer.

Still another object of the present invention is to use a pharmaceuticalcomposition of asiaticoside optionally along with acceptablepharmaceutical excipients, for treatment of Helicobacter Pylori.

Still another object of the present invention is to use a pharmaceuticalcomposition of asiaticoside optionally along with acceptablepharmaceutical excipients, for prevention of gastric diseases andgastric carcinoma.

STATEMENT OF THE INVENTION

The present invention discloses a commercially viable pharmaceuticalgrade asiaticoside composition extracted from a plant material centellaasiatica having at least 99% purity. The present invention discloses anin-vivo method of the treatment in humans and animals wherein the methodof treatment comprises oral administration of the asiaticosidecomposition adapted for treatment of Inflammatory Bowel Diseases,treatment of hemorrhoids, anal fissures, and fistulas, prevention of acolon ulcer and cancer, treatment of eradication of a helicobacterpylori infection, prevention of gastric diseases, and prevention of agastric carcinoma.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present invention, Asiaticoside compound disclosedherein has the following structure:

Asiaticoside:

The present disclosure relates to a process to obtain asiaticoside of atleast 99% purity, said process comprising acts of:

-   -   a) treating pulverized source material of asiaticoside with        first solvent;    -   b) extracting the treated pulverized material with a second        solvent to obtain an extract or filtrate;    -   c) distilling and dissolving the extract or the filtrate in a        third solvent to obtain a solution;    -   d) passing the solution through adsorbent and eluting with an        alcoholic solvent to obtain a solvent elute; and    -   e) vacuum concentrating the solvent elute to obtain a powder of        asiaticoside; and    -   f) dissolving the powder in alcohol and crystallizing to obtain        the asiaticoside of at least 99% purity.

The present disclosure also relates to a process of preparing apharmaceutical composition comprising asiaticoside of at least 99%purity along with pharmaceutically acceptable excipient(s), said processcomprising acts of:

-   -   a) treating pulverized source material of asiaticoside with        first solvent;    -   b) extracting the treated pulverized material with a second        solvent to obtain an extract or filtrate;    -   c) distilling and dissolving the extract or the filtrate in a        third solvent to obtain a solution;    -   d) passing the solution through adsorbents and eluting with an        alcoholic solvent to obtain a solvent elute;    -   e) vacuum concentrating the solvent elute to obtain a powder of        asiaticoside;    -   f) dissolving the powder in alcohol and crystallizing to obtain        asiaticoside; and    -   g) adding the pharmaceutically acceptable excipient(s) to the        asiaticoside to obtain the pharmaceutical composition comprising        asiaticoside of at least 99% purity.

In an embodiment of the present disclosure, source material ofasiaticoside is selected from group comprising plants and animals.

In another embodiment of the present disclosure, plant source isCentella asiatica.

In still another embodiment of the present disclosure, extracting iscarried out at temperature ranging from about 20° C. to about 30° C., attime period of about 8 hours to about 24 hours.

In yet another embodiment of the present disclosure, first solvent isselected from group comprising aliphatic compounds, ketones, alcohols,nitrites, esters, ethers or any combination thereof, preferablypetroleum ether or methyl isobutyl ketone.

In yet another embodiment of the present disclosure, second solvent isan aliphatic alcohol selected from group comprising methyl alcohol,ethyl alcohol, propyl alcohol, isopropyl alcohol and butyl alcoholeither alone or in combination with water in a ratio ranging from about60% to about 99%.

In yet another embodiment of the present disclosure, extract isdissolved in demineralized water.

In yet another embodiment of the present disclosure, third solvent isselected from group comprising hexane, methyl isobutyl ketone andpetroleum ether or any combination thereof.

In yet another embodiment of the present disclosure, adsorbent is aresin bed.

In yet another embodiment of the present disclosure, alcoholic solventhas carbon atom ranging from C-1 to C-4 selected from group comprisingmethanol and isopropyl alcohol or any combination thereof.

In yet another embodiment of the present disclosure, said vacuumconcentrating is carried out at temperature ranging from about 50° C. toabout 65° C.

In yet another embodiment of the present disclosure, aqueous alcohol isselected from group comprising ethyl alcohol, methyl alcohol, isopropylalcohol and any combination thereof.

In yet another embodiment of the present disclosure, crystallizing iscarried out at temperature ranging from about −15° C. to about 0° C.

In yet another embodiment of the present disclosure, composition isformulated into tablets, troches, lozenges, aqueous or oily suspensions,ointments, patches, gels, lotions, dentifrices, capsules, emulsions,creams, sprays, drops, dispersible powders or granules, emulsions inhard or soft gel capsules, syrups, elixirs, phytoceuticals,nutraceuticals and food stuffs or any combination thereof.

The present disclosure also relates to a method of treating a diseaseselected from group comprising inflammatory Bowel Disease, hemorrhoid,anal fissure, fistula, gastric disease, gastric carcinoma anderadication of a helicobacter pylori infection or any combinationthereof, said method comprises act of administering a dose ofasiaticoside optionally along with pharmaceutically acceptableexcipient(s) to a subject in need thereof.

The present disclosure also relates to a method of preventing a diseaseselected from group comprising colon cancer, gastric disease, gastriccarcinoma and ulcerogenic effect of drugs or any combination thereof,said method comprising act of administering a dose of asiaticosideoptionally along with pharmaceutically acceptable excipient(s) to asubject in need thereof.

In an embodiment of the present disclosure, pharmaceutically acceptableexcipient is selected from group comprising granulating agents, bindingagents, lubricating agents, disintegrating agents, sweetening agents,glidants, anti-adherents, anti-static agents, surfactants,anti-oxidants, gums, coating agents, colouring agents, flavouringagents, coating agents, plasticizers, preservatives, suspending agents,emulsifying agents and spheronization agents or any combination thereof.

In another embodiment of the present disclosure, subject is an animal,including human.

In still another embodiment of the present disclosure, composition isadministered to animals at dosage ranging from about 1 mg/kg to about360 mg/kg.

In yet another embodiment of the present disclosure, composition isadministered to humans at dosage ranging from about 1 mg/kg to about 60mg/kg.

In a preferred embodiment, the present invention provides a highly purecomposition of asiaticoside having at least 99% purity and a process ofmanufacturing thereof. Asiaticoside of the present invention has amolecular formula C₄₈H₇₈O₁₉ Asiaticoside is obtained from plant sourceCentella asiatica in this one preferred embodiment, however, it isunderstood that Asiaticoside can be obtained from animal sources inother alternative embodiments of the present invention.

In particular, the present invention provides a method or process forpreparation of a composition having 99% or more of asiaticoside, whereinsaid process comprising steps of:

-   -   a) Pulverizing plant material of Centella asiatica    -   b) Treating pulverized material with a first solvent to remove        fatty substances, chlorophyll and other colorants;    -   c) Extracting the bioactive molecules using a second solvent;    -   d) Distilling extract to remove solvents and form a paste;    -   e) Dissolving distilled paste in a third solvent to obtain a        solution;    -   f) Extracting with a third solvent to remove acidic impurities;    -   g) Passing purified extract through adsorbents to get a clear        solution;    -   h) Eluting the resin bed with an alcoholic solvent to obtain a        solvent elute;    -   i) Vacuum concentration of the solvent elute to obtain a powder;        and    -   j) Aqueous-alcohol dissolution of the powder and crystallization        to obtain asiaticoside composition that has at least 99% HPLC        purity.

The detailed method or process for preparation of the 99% or more ofasiaticoside composition is described hereinafter:

In accordance with the present invention, dry leaves of Centellaasiatica including stems are pulverized to 40 mesh size. The pulverizedmaterial is packed in an extractor having perforated plates at the topand bottom of the vessel that is fitted with filter cloths.

In next step, a first solvent is circulated through the extractor in acounter current manner at temperature ranging between 20° C. to 30° C.,most preferably at 30° C., for a time period ranging between 8 hrs to 24hrs, most preferably for 12 hrs. In this one preferred embodiment thefirst solvent used is petroleum ether having a boiling range of 60° C.to 80° C. However, it is understood that the first solvent can beselected from a group comprising aliphatic compounds, ketones, alcohols,nitrites, esters, ether and mixtures of one or more thereof in otherembodiment of the present invention. In next step, Chlorophyll and otherlipids are removed from the extract and the mass is allowed dry free ofsolvent.

In next step, the dried mass is extracted in a counter current mannerusing a second solvent. The second solvent in this one preferredembodiment is a combination of aliphatic alcohols that has Carbon atomranging from 1 to 4, both straight chain and branched chain alcohols, incombination with or without water. In other alternative embodiments ofthe present invention, the second solvent can be selected from a methylalcohol, an ethyl alcohol, a propyl alcohol, an isopropyl alcohol and abutyl alcohol-either alone or in combination with water in a ratioranging from 60% to 99%.

In next step, the clear extract is distilled under reduced pressure toevaporate solvent and to form a paste. In next step, the paste isdissolved in demineralized water and filtered clear of insolubles. Innext step, the clear water layer is extracted repeatedly and washed witha third solvent to get rid of acidic material and to obtain a clearaqueous extract. The third solvent in this one preferred embodiment ismethyl isobutyl ketone. In still another embodiment of the presentinvention, the solvent can be selected from a group comprising hexaneand petroleum ether.

In next step, the clear aqueous extract layer is adapted to be passedthrough a bed of adsorbent grade resin and washed to get rid of all thecolors and contaminants out of the bed with 5 volumes or more preferably8 volumes of deionised water.

In next step, the water washed bed is eluted with an alcoholic solventhaving carbon atom ranging from C-1 to C-4, preferably methanol orisopropyl alcohol or a mixture of the said alcohols. The eluted solventis collected and the bed repeatedly washed to get all thecentellosaponins out of the bed.

In next step, the solvent elute is concentrated under vacuum at lowtemperature preferably between 50° C. to 65° C. to a powder. The powderis redissolved in aqueous alcohol to form a solution. The aqueousalcohol used in this one preferred embodiment is methyl alcohol.However, it is understood that aqueous alcohol can be selected from agroup of ethyl, propyl alcohol or isopropyl alcohol—either alone or incombination with water in ratios ranging from 50% to 99% of alcohol.

In next step, the solution is cooled to less than 0° C., preferably at−15° C. or below to crystallize the asiaticoside. In next step, the pureasiaticoside crystals obtained is filtered off and washed with coldwater until free of salts and dried under vacuum to get free flowingpowder that can be analyzed for its purity using HPLC for assay.

A summary of the most preferred process is as follows:

-   -   a) Dry leaves of Centella asiatica including stems were        pulverized to 40 mesh size.    -   b) Pulverized material is packed in an extractor having        perforated plates at top and bottom of the vessel having filter        cloths.    -   c) Petroleum ether having a boiling range of 60° C. to 80° C. is        circulated in a counter current manner at temperature ranging        between 20° C. to 30° C. preferably at 30° C. for a time period        ranging between 8 hrs to 24 hrs preferably for 12 hrs.    -   d) The resultant extract consisting of Chlorophyll and other        lipids are removed and the mass allowed dry free of solvent.    -   e) The dried mass is extracted again in a counter current manner        using a combination of aliphatic alcohol solvents having Carbon        atom ranging from 1 to 4 both straight chain and branched chain        alcohols in combination with or without water. The said alcohols        can be methyl, ethyl, propyl, isopropyl, butyl alcohol either        alone or in combination with water in the ratio ranging from 60%        to 99% of said alcohol.    -   f) The clear extract is made into a paste by evaporating solvent        by distillation under reduced pressure.    -   g) The paste is dissolved in demineralized water and washed        repeatedly with methyl isobutyl ketone.    -   h) The clear aqueous extract is then passed through a bed of        adsorbent grade resin and washed free of all the colors and        contaminants out of the bed with 5 volumes or more preferably 8        volumes of deionised water.    -   i) The water washed bed was eluted with an alcoholic solvent        having carbon atom ranging from C-1 to C-4, preferably methanol        or isopropyl alcohol or a mixture of the said alcohols.    -   j) The eluted solvent was collected and the bed repeatedly        washed to get all the centellosaponins out of the bed.    -   k) The solvent elute was concentrated under vacuum at low        temperature preferably between 50° C. to 65° C. to a powder.    -   l) The powder is redissolved in aqueous alcohol in which the        alcohol used is ethyl, methyl, propyl or isopropyl in        combination or with water in ratios ranging from 50% to 99% of        alcohol and cooled to less than 0° C. preferably at −15° C. or        below to crystallize the asiaticoside.    -   m) The pure asiaticoside thus formed is filtered off and washed        with cold water until free of salts and dried under vacuum to        get free flowing powder.    -   n) The material thus isolated is analyzed for its purity in the        following manner using HPLC for assay as well as impurities.

HPLC method for analysis is as follows:

Column: 250 mm × 4.6 mm Reverse phase C-18 particle size 5μ Wavelengthof detector: 220 nm Flowrate: 1.4 ml/min Standard Used: Chromadex Lot No11030-291 Time Acetonitrile Water Initial 75% 25% 30 mins 45% 55% 40mins 75% 25%

In still another embodiment of the present invention, Asiaticosidecomposition is optionally usable along with acceptable pharmaceuticalexcipients selected from a group such as but not limited to granulatingagents, binding agents, lubricating agents, disintegrating agents,sweetening agents, glidants, anti-adherents, anti-static agents,surfactants, anti-oxidants, gums, coating agents, coloring agents,flavouring agents, coating agents, plasticizers, preservatives,suspending agents, emulsifying agents and spheronization agents.

In still another embodiment of the present invention, Asiaticosidecomposition can be formulated into various dosage forms selected from agroup such as tablets, troches, lozenges, aqueous or oily suspensions,ointments, patches, gels, lotions, dentifrices, capsules, emulsions,creams, sprays, drops, dispersible powders or granules, emulsions inhard or soft gel capsules, syrups, elixirs, phytoceuticals,nutraceuticals and food stuffs, for example.

In still another embodiment of the present invention, composition iseither a powder or liquid and has minimal side effects, wherein thecomposition is in a dosage range of 1-360 mg/kg in animals and 1-60mg/kg in human beings. The asiaticoside composition adapted to beadministered as a therapeutic or a prophylactic dose for effectivetreatment of a diseased site.

In the preferred embodiment, the present invention additionally providesan in-vivo method of treating Inflammatory Bowel Diseases (IBDhereinafter) such as Ulcerative Colitis (UC hereinafter), Crohn'sDisease (CD hereinafter) and associated complications of hemorrhoids,anal fissures, fistulas, and prevention of colon cancer induced by theabove conditions.

In the preferred embodiment, the present invention also provides anin-vivo method of treating Helicobacter Pylori (H. Pylori herein after).Moreover, the present invention also provides an in-vivo method oferadication of H. Pylori in NSAID induced ulcers.

In the preferred embodiment, the present invention also provides anin-vivo method of preventing gastric diseases and gastric carcinoma.

Accordingly, in the context of the present invention, the tests that areconducted to ascertain an anti-inflammatory activity, a healingactivity, a method of use and an establishment of mode of action of theasiaticoside composition (test drug hereinafter) are described in detailhereinafter:

-   -   a) In a pharmacokinetic study, the test drug is administered and        its bioavailability in blood and excretion in urine and feces        are analyzed. It is observed that asiaticoside not absorbs into        the blood, but excretes completely in the fecal matter in the        unchanged form. This property of the test drug prompted further        investigation for treating IBD and related diseases.    -   b) In trinitrobenzene sulfonic acid (TNBS) induced IBD model,        the test drug on post treatment for 10 days after TNBS        administration has significantly reduced colon weight and colon        weight/length ratio. In addition, increased body weight,        decreased ulcer index and myeloperoxidase activity in colon is        seen. Also, the rectal bleeding is significantly reduced after        3^(rd) day of test drug administration.    -   c) In acetic acid induced IBD model, the test drug after 10 days        of treatment significantly reduced colon weight, colon length,        colon width, colon weight/length ratio and macroscopic score of        colon. The test drug also showed decreased ulcer index and        significant reduction in myeloperoxidase activity.    -   d) In-vivo model of H. Pylori infected rats with non steroidal        anti inflammatory drug (NSAID hereinafter) induced ulcers is        tested for the potential anti-H. pylori and anti-ulcer activity        of the test drug. The test drug has showed dose dependent and        time dependent reduction in the ulcer area along with complete        eradication of H. pylori infection.    -   e) In a model of stress induced ulcers, histamine is        administered to rats to induce ulcerative conditions and the        effect of the test drug in preventing stress induced ulcer is        confirmed by pretreating the animal with the test drug. The test        drug is found effective in preventing histamine induced ulcers.    -   f) In a model of alcohol induced ulcers, ethanol is administered        to rats that are pretreated with the test drug to induce        ulcerative conditions. The test drug has showed prophylactic        effect against ethanol induced ulcers.

The invention is further elaborated with the help of following examples.However, it is understood that these examples should not be construed tolimit the scope of the invention.

EXAMPLES Example-1

10 kilograms of Centella asiatica leaves were pulverized to 40 mesh sizeusing hammer mill. The pulverized material was extracted with 50 litersof petroleum ether in a fixed bed counter current extractor repeatedlyover a period of 8 hrs at 30° C. The extracted mass free of chlorophylland fats was dried by circulating air into the bed. The dried mass isextracted with 75 liters of isopropyl alcohol at 30° C. for 8 hrs. Theclear filtrate after extraction was concentrated under vacuum to get apaste. The paste was dissolved in 25 liters of demineralized water andfiltered clear of insolubles. The clear water layer was extractedrepeatedly and washed with methyl isobutyl ketone to get rid of allacidic material. This operation was monitored for absence of acidiccomponents using TLC wherein TLC mobile phase was consisting of toplayer of mixture n-butanol:ethyl acetate:water in the ratio of 4:1:5.The clear layer was passed through an adsorbent chromatographic columnhaving vertical Column containing 250 ml of Amberlite® XAD-4 and washedwith water to get rid of all adhering colours and particles. This wasfollowed by elution cycle wherein the column was eluted with methylalcohol and the solvent elute was concentrated under vacuum to get 500gms powder. This powder was dissolved in 2500 ml of an aqueous alcoholcomprising of 80% methyl alcohol and 20% water to get a clear solution.This solution was cooled to −15° C. to get asiaticoside crystals. Themass was filtered, water washed and dried at 80° C. The yield was 158gms of asiaticoside having HPLC purity 99.6%

Example-2

10 kilograms of Centella asiatica leaves were pulverized to 40 mesh sizeusing hammer mill. The pulverized material was extracted with 50 litersof petroleum ether in a fixed bed counter current extractor repeatedlyover a period of 8 hrs at 30° C. The extracted mass free of chlorophylland fats was dried by circulating air into the bed. The dried mass wasextracted with 75 litres of 80% aqueous ethyl alcohol at 30° C. for 8hrs. The clear filtrate, after extraction, was concentrated under vacuumto get a paste. The paste was dissolved in 25 liters of demineralizedwater and filtered clear of insolubles. The clear water layer wasextracted repeatedly and washed with methyl isobutyl ketone to get ridof all acidic material. This operation was monitored for absence ofacidic components using TLC wherein the TLC mobile phase was consistingof top layer of a mixture having n-butanol:ethyl acetate:water in theratio of 4:1:5. The clear layer was passed through an adsorbentchromatographic column having vertical Column containing 250 ml ofAmberlite® XAD-4 and washed with water to get rid of all adheringcolours and particles. This was followed by elution cycle wherein thecolumn was eluted with methyl alcohol and the solvent elute obtained isconcentrated under vacuum to get 500 gms powder. The powder wasdissolved in 2500 ml of an aqueous alcohol comprising of 80% methylalcohol and 20% water to get a clear solution. This solution was cooledto −15° C. to get asiaticoside crystals. The mass was filtered, waterwashed and dried at 80° C. The yield obtained was 150 gms ofasiaticoside having HPLC purity 99.2%

Example-3

10 kilograms of Centella asiatica leaves were pulverized to 40 mesh sizeusing hammer mill. The pulverized material was extracted with 50 litersof petroleum ether in the fixed bed counter current extractor repeatedlyover a period of 8 hrs at 30° C. The extracted mass free of chlorophylland fats was dried by circulating air in to the bed. The dried mass wasextracted with 75 liters of methyl alcohol at 30° C. for 8 hrs. Theclear filtrate obtained after extraction was concentrated under vacuumto get a paste. The paste was dissolved in 25 liters of demineralizedwater and filtered clear of insolubles. The clear water layer wasextracted repeatedly and washed with methyl isobutyl ketone to get ridof acidic material. The operation for absence of acidic components wasmonitored using TLC. The clear layer was passed through an adsorbentchromatographic column having vertical Column containing 250 ml ofAmberlite® XAD-4 and washed with water to get rid of all adheringcolours and particles. This was followed by an elution cycle wherein thecolumn was eluted with methyl alcohol to obtain a solvent elute. Thesolvent elute is concentrated under vacuum to get 500 gms powder. Thispowder was dissolved in 2500 ml of an aqueous alcohol, comprising of 80%methyl alcohol and 20% water, to get a clear solution. This solution wascooled to −15° C. to get Asiaticoside crystals. The mass was filtered,water washed, and dried at 80° C. The yield obtained was 145 gms ofAsiaticoside having HPLC purity 99.8%

Example-4

10 kilograms of Centella asiatica leaves were pulverized to 40 mesh sizeusing hammer mill. The pulverized material was extracted with 50 litersof petroleum ether in a fixed bed counter current extractor repeatedlyover a period of 8 hrs at 30° C. The extracted mass free of chlorophylland fats was dried by circulating air into the bed. The dried mass isextracted with 75 liters of Isopropyl alcohol, containing 80% isopropylalcohol and 20% water, at 30° C. for 8 hrs. The clear filtrate afterextraction was concentrated under vacuum to get a paste. The paste wasdissolved in 25 liters of demineralized water and filtered clear ofinsolubles. The clear water layer was extracted repeatedly and washedwith methyl isobutyl ketone to get rid of all acidic material. Thisoperation was monitored for absence of acidic components using TLC. Theclear layer was passed through an adsorbent chromatographic columnhaving vertical column containing 250 ml of Amberlite® XAD-4 and washedwith water to get rid of all adhering colours and particles. This wasfollowed by an elution cycle wherein the column was eluted with methylalcohol and the solvent elute obtained is concentrated under vacuum toget 500 gms powder. The powder was dissolved in 2500 ml of an aqueousalcohol, comprising of 80% methyl alcohol and 20% water, to get a clearsolution. The clear solution was cooled to −15° C. to get asiaticosidecrystals. The asiaticoside mass was filtered, water washed and dried at80° C. The yield was 158 gms of asiaticoside having HPLC purity 99.6%

Example-5

10 kilograms of Centella asiatica leaves were pulverized to 40 mesh sizeusing a hammer mill. The pulverized material was extracted with 50liters of petroleum ether in a fixed bed counter current extractorrepeatedly over a period of 8 hrs at 30° C. The extracted mass free ofchlorophyll and fats was dried by circulating air in to the bed. Thedried mass is extracted with 75 liters of 70% isopropyl alcoholcontaining 30% water at 30° C. for 8 hrs. The clear filtrate afterextraction was concentrated under vacuum to get a paste. The paste wasdissolved in 25 liters of demineralized water and filtered clear ofinsolubles. The clear water layer was extracted repeatedly and washedwith methyl isobutyl ketone to get rid of all acidic material. Thisoperation was monitored for absence of acidic components using TLC. Theclear layer was passed through an adsorbent chromatographic columnhaving vertical column containing 250 ml of Amberlite® XAD-4 and washedwith water to get rid of all adhering colours and particles. This wasfollowed by elution cycle wherein the column was eluted with methylalcohol and the solvent elute obtained is concentrated under vacuum toget 500 gms powder. This powder was dissolved in 2500 ml of an aqueousalcohol, comprising of 80% methyl alcohol and 20% water, to get a clearsolution. The clear solution was cooled to −15° C. to get asiaticosidecrystals. The mass was filtered, water washed and dried at 80° C. Theyield obtained was 157 gms of asiaticoside having HPLC purity 99.8%.

Example-6 Availability of Test Drug at Site of Action

The Pharmacokinetic parameters of the test drug were studied in healthyrats to determine the bioavailability of the test drug.

Procedure: In this study, animals were fasted overnight before theexperiment and were given a single oral dose of 250 mg/kg of test drug.The blood samples were collected at 0, 0.25, 0.50, 1, 2, 3, 4, 5 and 6hours after dosing. Plasma was obtained by centrifugation of blood at7000 rpm for 15 min at 4° C. and kept at −20° C. until analysis wascarried out.

The animals were kept in the metabolic cages for collection of urine andfeces. A Reverse Phase HPLC method was developed for detection of thetest drug in the plasma, urine and feces. Elimination of the test drugby urine and feces was determined at 0-4 hr, 4-8 hr and 8-24 hr.

Observation: It was observed that no drug was absorbed into the bloodsystemically even at the high dose of 250 mg/kg. This was reconfirmed byabsence of asiaticoside in urine. It was observed that the test drugpasses in an unchanged form through the gastro intestinal tract andcompletely excretes through the fecal matter. Further, it was observedthat the test drug does not absorb into the blood that substantiallyincreases the bioavailability of the asiaticoside composition at adiseased site. In the experiments conducted in this context, it wasobserved that about 81.42% of the drug was recovered from fecal matter.Hence, it was confirmed that there is a potential for the test drug intreatment of IBD and related diseases.

Example-7 Effect of Test Drug in TNBS Induced Colitis in Rats

The trinitrobenzenesulfonic acid (TNBS) induced colitis is one of thestandard animal models used for IBD. In this model, a singleintracolonic administration of TNBS damages the colonic epithelium andinduces prolonged chronic inflammation. The transmural granulomatousinflammation of the colon is similar to histopathological featuresexhibited in Crohn's disease.

Procedure: In this study, Male Wistar rats (280-291 g) were fasted for48 hrs, and put under anaesthesia. A 3 mm diameter cannula was insertedinto the anus to the depth of 8 cm and 25 mg of TNBS dissolved in 0.25ml of 30% ethanol solution was injected to each rat. The rats werepositioned tail up for 1 minute. After 1 day, the test drug wasadministered orally once daily for 10 days. A healthy control groupreceiving only saline but no TNBS was maintained. On day 11, each groupof rats were sacrificed by cervical dislocation and the colon wasisolated for physical examination. Colon samples were preserved in 10%neutral formalin solution for histopathological evaluation.

Observations: In the performed experiments, TNBS induced IBD conditionssuch as increased colon weight, colon weight to length ratio andmyloperoxidase activity were reversed significantly by treatment withtest drug for 10 days at a dose of 50 mg/kg (p.o.) once daily. The testdrug also healed the lesions produced by TNBS as seen from the reductionin microscopic scores and colonic ulcer index. The reversal ofmyeloperoxidase activity by test drug confirmed its potential in healingcolonic lesions seen in IBD.

TABLE 1 EFFECT OF TEST DRUG ON COLON WEIGHT AND LENGTH IN TNBS INDUCEDIBD IN RATS AFTER 10 DAYS OF TREATMENT Healthy control TNBS control TestDrug Colon 1.15 ± 0.02 2.22 ± 0.1^(###)   1.71 ± 0.07*** weight (g)Colon 17.2 ± 0.53 13.64 ± 0.34^(###)  14.14 ± 0.53   length (mm) Colon0.377 ± 0.32  0.99 ± 0.88^(###) 0.73 ± 0.13  width (mm) Colon 0.00 ±0.00 8.80 ± 0.58^(###) 4.40 ± 0.68** weight/ length ratio Myelo-  0.01 ±0.0002  0.05 ± 0.0003^(###)   0.02 ± 0.0003*** peroxi- dase activity(U/mg) n = 6; For each of the parameters, data analyzed using One WayANOVA followed by Dunnett test. ^(###)P < 0.001, as compared to Healthycontrol group; *P < 0.05, **P < 0.01 and ***P < 0.001 as compared toTNBS control group.

TABLE 2 EFFECT OF TEST DRUG ON COLON MICROSCOPIC SCORES IN TNBS INDUCEDIBD IN RATS AFTER 10 DAYS OF TREATMENT Healthy control TNBS control TestDrug Microscopic Score 0.00 ± 0.00 8.80 ± 0.58^(###) 4.40 ± 0.68***Colon Ulcer Index 0.00 ± 0.00 9.92 ± 0.31^(###) 2.85 ± 0.17*** n = 6;For each of the parameters, data analyzed using Kruskal Wallis followedby Dunns Multiple Comparison Test. ^(###)P < 0.001, as compared toHealthy control group; *P < 0.05, **P < 0.01 and ***P < 0.001 ascompared to TNBS control group.

TABLE 3 EFFECT OF TEST DRUG ON BODY WEIGHT IN TNBS INDUCED IBD IN RATSAFTER 10 DAYS OF TREATMENT Days Healthy control TNBS control Test DrugNormal 291.00 ± 1.83 280.33 ± 2.62^(ns)  286.50 ± 3.14^(ns1) (Day −2)Day 0 264.17 ± 2.21 250.00 ± 2.00^(# ) 252.67 ± 3.12^(ns1) Day 11 (10296.33 ± 1.45 229.60 ± 0.81^(###)  293.00 ± 0.84*** days of treatment) n= 6; Data analyzed using Two Way ANOVA followed by Bonferroni posttests. ^(#)P < 0.05, ^(###)P < 0.001 as compared to Healthy controlgroup; ***P < 0.001 as compared to TNBS control group on respective day.^(ns)Not significant as compared with Healthy control; ^(ns1)Notsignificant as compared with TNBS induced rats.

The effects of test drugs on TNBS induced IBD strongly indicatedtherapeutic value of test drug in treatment and management of IBD.

Example 8 Effect of Test Drug on Acetic Acid Induced Colitis in Rats

The acetic-acid induced colitis is a model of IBD with a similarity ofthe inflammatory mediators to that of acute human intestinalinflammation.

Procedure: In this model, initial injury was an epithelial necrosis andedema that variably extended into the lamina propria, submucosa, orexternal muscle layers, depending of the concentrations and length ofexposure to acetic acid. Male Wistar rats (280-291 g) were given thetest drug once daily at dose of 50 mg/kg (p.o.) starting 72 h beforeinduction of colitis. Rats were fasted for 24 hrs, anesthetized and acannula that has 3 mm diameter was inserted into the anus to the depthof 8 cm for instilling 2 ml of 3% acetic acid into the colon for 30 s.After 24 hrs, animals were sacrificed by cervical dislocation, the colonwas isolated and the parameters of colitis were measured.

Observations: Pre-treatment with the test drug in rats for 3 days had asignificant reversal effect in acetic acid induced colitis symptoms. Thereversal of acetic acid induced conditions such as increased colonweight and colon weight to length ratio, which indicated beneficialeffects of test drugs on colitis. These effects were further confirmedby decrease in macroscopic lesion score and myeloperoxidase activity incolon tissues.

TABLE 4 EFFECT OF TEST DRUG ON COLON PARAMETERS IN ACETIC ACID INDUCEDCOLITIS IN RATS FOLLOWING PRETREATMENT FOR 3 DAYS Acetic acid Healthycontrol control Test Drug Colon weight 1.11 ± 0.07 2.49 ± 0.14^(###)1.53 ± 0.10*** (g) Colon length 17.73 ± 0.18  13.55 ± 0.44^(###)  16.22± 0.63***  (mm) Colon width 0.40 ± 0.03 1.07 ± 0.06^(###) 0.71 ± 0.05***(mm) Colon weight/  0.06 ± 0.0002 0.18 ± 0.006^(#)  0.09 ± 0.01***length ratio Myeloperoxi-  0.01 ± 0.001  0.04 ± 0.006^(###) 0.02 ±0.002** dase activity (U/mg) n = 6; For each of the parameters, dataanalyzed using One Way ANOVA followed by Dunnett test. ^(###)P < 0.001,as compared to Healthy control group; ***P < 0.001 as compared to AceticAcid control group.

TABLE 5 EFFECT OF TEST DRUG ON COLON MICROSCOPIC SCORES IN ACETIC ACIDINDUCED COLITIS IN RATS FOLLOWING PRETREATMENT FOR 3 DAYS Acetic AcidHealthy control control Test Drug Microscopic score 0.00 ± 0.00  9.33 ±0.33^(###) 4.33 ± 0.71**  Colon ulcer Index 0.00 ± 0.00 13.72 ±0.48^(###) 4.34 ± 0.29*** n = 6; For each of the parameters, dataanalyzed using Kruskal Wallis followed by Dunns Multiple ComparisonTest. ^(###)P < 0.001, as compared to Healthy control group; **P < 0.01and ***P < 0.001 as compared to Acetic Acid control group.

The effects of test drugs on acetic acid induced colitis stronglyindicated therapeutic value of test drug in management of IBD.

Example 9 Effect of Test Drug on H. Pylori Infected Gastric Diseases

The effect of test drug on eradication of H. Pylori was tested in NSAIDinduced ulcers in rats after chronic administration for 9 weeks. H.Pylori infection was monitored by Polymerase Chain Reaction (PCR) andRapid Urease Test (RUT) to validate the chronic treatment.

Procedure: Male Wistar rats (200-230 g) were fasted for 24 hrs and givenNaproxen at a dose of 30 mg/kg (p.o.) for 3 consecutive days. After 24hrs, brucella broth solution of viable H. pylori (ATCC 26695, strainMS-5, 10⁸ CFU) was administered (1 ml/animal) consecutively for 3 days.After 1 week, the test drug was administered at doses of either 120mg/kg (p.o.) twice daily or 240 mg/kg (p.o.) once daily or 360 mg/kg(p.o.) once daily. The animals were sacrificed on completion of 9 weeksof treatment. Stomach of the sacrificed animal was isolated, gastricmucosa washed with saline and the pylorus was dissected for RUT. Mucosawas scrapped for myeloperoxidase estimation and DNA was amplified byPCR, gel electrophoresis for two non mutating genes 16S rRNA and hrgA.

Observations: The infection status was monitored in the stomach of ratsby RUT and PCR amplification of two H. pylori genes after 4, 6 and 9weeks of treatment. It was observed that the test drug at a dose of 360mg/kg (p.o.) shows rapid antibacterial action on 3^(rd) week andsubsequent eradication of H. Pylori infection in 9 weeks. However, thetest drug administered at lower doses of 120 mg/kg (p.o.) twice dailyand 240 mg/kg (p.o.) once daily was not effective in H. Pylorieradication. The effect of the test drug strongly indicated atherapeutic value in eradication of H. Pylori infection at high dosesand also provided a pharmacological credence to the therapeuticpotential for treatment of H. Pylori related gastric diseases andgastric carcinomas.

Example 10 Prophylactic Effect of Test Drug on Drug Induced GastricDiseases

Gastric lesions induced by NSAIDS drugs such as Naproxen can aggravateinflammatory reactions in the GI tract. The potential of the test drugin preventing drug induced gastric lesions was studied in animal modelsof Naproxen induced ulcers.

Procedure: Male Wistar rats (220-230 g) were pretreated with the testdrug at doses of either 30 mg/kg (p.o.) or 60 mg/kg (p.o.) or 120 mg/kg(p.o.) and then Naproxen was administered at a dose of 30 mg/kg (p.o.).The formation of ulcer and area of ulceration were compared with theNaproxen control group that did not undergo pretreatment with the testdrug.

Observations: It was observed that pretreatment with the test drugprevented ulcer formation in a dose dependent manner. The highest doseof 120 mg/kg was found to be most effective in protecting againstulcerogenic action of Naproxen. Accordingly, the test showed potentialprophylactic effect against ulcerogenic action of drugs.

TABLE 6 PROPHYLACTIC EFFECT OF TEST DRUG ON NAPROXEN INDUCED GASTRICDISEASES IN RATS Area of Ulceration (mm²) Naproxen Control 5.50 ± 0.158 Test Drug (30 mg/kg) + Naproxen 5.49 ± 0.212^(ns)  Test Drug (60mg/kg) + Naproxen 4.52 ± 0.169** Test Drug (120 mg/kg) + Naproxen 2.632± 0.146*** n = 6; Data analyzed using One Way ANOVA followed byDunnett's Multiple Comparison test. ^(ns)Not significant; **P < 0.01,***P < 0.001 as compared to Naproxen control group.

Example 11 Prophylactic Effect of Test Drug on Stress Induced GastricDiseases

Stress induced gastric lesion is a condition that can cause or aggravateinflammatory reactions in the GI tract. The potential of the test drugin preventing stress induced gastric lesions was studied in the animalmodels of histamine induced ulcers.

Procedure: Male Wistar rats (220-230 g) were pretreated with the testdrug at the doses of either 30 mg/kg (p.o.) or 60 mg/kg (p.o.) or 120mg/kg (p.o.) and then Histamine was administered at a dose of 300 mg/kg(i.p.). The formation of ulcer and area of ulceration were compared witha control group which did not undergo pretreatment with test drug.

Observations: It was observed that pretreatment with the test drugprevented the ulcer formation in a dose dependent manner. The highestdose of 120 mg/kg was most effective in protecting against ulcerogenicaction of Histamine. Hence, it was confirmed that the test drug haspotential prophylactic effect against stress induced ulcers.

TABLE 7 PROPHYLACTIC EFFECT OF TEST DRUG ON HISTAMINE INDUCED GASTRICDISEASES IN RATS Area of Ulceration (mm²) Histamine Control 10.66 ±0.1302  Test Drug (30 mg/kg) + Naproxen 9.242 ± 0.415*  Test Drug (60mg/kg) + Naproxen 7.062 ± 0.362*** Test Drug (120 mg/kg) + Naproxen4.598 ± 0.323*** n = 6; Data analyzed using One Way ANOVA followed byDunnett's Multiple Comparison test. *P < 0.05, ***P < 0.001 as comparedto Histamine control group.

Example 12 Prophylactic Effect of Test Drug on Alcohol Induced GastricDiseases in Rat

Consumption of alcohol is another major factor resulting in gastriclesions. Accordingly, the potential of the test drug in preventingethanol induced ulcers in animals was studied.

Procedure: Male Wistar rats were pretreated with the test drug at dosesof either 30 mg/kg (p.o.) or 60 mg/kg (p.o.) or 120 mg/kg (p.o.) andthen Ethanol was administered at a dose of 8 ml/kg (p.o.). The formationof ulcer and area of ulceration were compared with a control group whichdid not undergo pretreatment with the test drug. The mean ulcer area inthis study was measured to be 174.4 mm²±5.814 mm² in the control groupindicating the ulcerogenic effect of ethanol.

Observations: It was observed that pretreatment with the test drug hasprevented ulcers formation in a dose dependent manner. The highest doseof 120 mg/kg was most effective in protecting against ulcerogenic actionof Ethanol. Accordingly, the significant prophylactic effect of the testdrug against ulcers induced by alcohol consumption was confirmed.

TABLE 8 PROPHYLACTIC EFFECT OF TEST DRUG ON ETHANOL INDUCED GASTRICDISEASES IN RATS Area of Ulceration (mm²) Ethanol Control 174.4 ±5.814   Test Drug (30 mg/kg) + Naproxen 159.3 ± 4.846*  Test Drug (60mg/kg) + Naproxen 137.6 ± 4.069*** Test Drug (120 mg/kg) + Naproxen110.4 ± 2.305*** n = 6; Data analyzed using One Way ANOVA followed byDunnett's Multiple Comparison test. *P < 0.05, ***P < 0.001 as comparedto Ethanol control group.

Example 13 Effect of Test Drug in Treatment of IBD in Humans

A prospective study to assess the efficacy of the test drug in patientswith IBD was conducted.

Procedure: In this study, three patients suffering from chronic symptomsof inflammatory bowel disease were given the test drug at a dose of 300mg twice daily for a period of 6 months. Thereafter, the efficacy of thetest drug was analyzed on the basis of patient reported outcome taken atthe beginning and end of the study period.

Observations: It was observed that all patients were experiencing relieffrom IBD related symptoms such as abdominal pain, diarrhea, rectalbleeding, fever, nausea and vomiting. It was further observed that therewas significant improvement in bowel control which was seen from reducedfrequency of visits to restrooms, dependence on diarrhoea medication andpain associated with bowel movement. It was further observed that therewere no incidences of joint pain, skin diseases or eye relatedcomplications. It was further observed that the test drug was welltolerated and showed efficacy in treatment of IBD.

TABLE 9 EFFECT OF TEST DRUG IN TREATMENT OF IBD IN HUMANS FOR 6 MONTHSPatient Recorded Outcome† Patient 1 Patient 2 Patient 3 Symptoms BeforeAfter Before After Before After Abdominal pain 3 1 3 0 3 1 Diarrhea 3 03 0 3 2 Bleeding 3 0 3 0 3 0 Frequent trips 3 0 3 1 3 0 to the bathroomDependence on 3 0 3 0 3 1 medication diarrhea and pain †Scale ofSeverity of IBD symptoms (0—Absence; 1—Mild; 2—Moderate; 3—Severe)

Example 14 Effect on Test Drug in Treatment of Hemorrhoids in Humans

A prospective uncontrolled pilot study to access the efficacy of thetest drug in patients with hemorrhoids was conducted.

Procedure: In this study, five patients suffering from chronichaemorrhoid symptoms of rectal bleeding, pain during defecation andrectal irritation were given the test drug at a dose of 300 mg twicedaily for 15 days. The efficacy of the test drug was analyzed on thebasis of patient reported outcome taken at the beginning and end of thestudy period.

Observations: It was observed that the test drug had an efficacy inproviding immediate relief from symptoms of Haemorrhoids and analfissures. It was further observed that all patients were relieved ofrectal bleeding on the 3^(rd) day of treatment with the test drug. Itwas further observed that there was significant relief in pain andirritation associated with bowel movement by the end of the treatmentperiod.

TABLE 10 EFFECT OF TEST DRUG IN TREATMENT OF HEMORRHOIDS IN HUMANS FOR15 DAYS Median Value of Patient Reported Outcome† (n = 5) SymptomsBefore Treatment After Treatment Pain 5 2 Edema 3 1 Itching 5 2 Bloodystool 5 0 Difficulty to 5 1 sit or walk †Scale of assessment (0 -Absence to 5 - Severe)

The present invention has been described in an illustrative manner, andit is to be understood that the terminology used is intended to be inthe nature of description rather than of limitation.

It is not intended to be exhaustive or to limit the invention to theprecise form disclosed. Many modifications and verifications arepossible in light of the above teaching. It is intended that the scopeof the invention be limited not by this detailed description, but ratherby the claims appended hereto. It is also to be understood that thefollowing claims are intended to cover all of the generic and specificfeatures of the invention described herein.

The invention claimed is:
 1. A method of treating a disease selectedfrom the group comprising inflammatory Bowel Disease, hemorrhoid, analfissure, fistula, gastric disease, gastric carcinoma and eradication ofa helicobacter pylori infection or any combination thereof, said methodcomprising administering a dose of asiaticoside optionally along withpharmaceutically acceptable excipient(s) to a subject in need thereof.2. The method as claimed in claim 1, wherein said pharmaceuticallyacceptable excipient is selected from the group comprising granulatingagents, binding agents, lubricating agents, disintegrating agents,sweetening agents, glidants, anti-adherents, anti-static agents,surfactants, anti-oxidants, gums, coating agents, colouring agents,flavouring agents, plasticizers, preservatives, suspending agents,emulsifying agents and spheronization agents, or any combinationthereof.
 3. The method as claimed in claim 1, wherein said subject is ananimal, including a human.
 4. The method as claimed in claim 1, whereinsaid composition is administered to animals at a dosage ranging fromabout 1 mg/kg to about 360 mg/kg.
 5. The method as claimed in claim 1,wherein said composition is administered to humans at a dosage rangingfrom about 1 mg/kg to about 60 mg/kg.
 6. A method of preventing adisease selected from the group comprising colon cancer, gastricdisease, gastric carcinoma and ulcerogenic effect of drugs or anycombination thereof, said method comprising administering a dose ofasiaticoside optionally along with pharmaceutically acceptableexcipient(s) to a subject in need thereof.
 7. The method as claimed inclaim 6, wherein said pharmaceutically acceptable excipient is selectedfrom the group comprising granulating agents, binding agents,lubricating agents, disintegrating agents, sweetening agents, glidants,anti-adherents, anti-static agents, surfactants, anti-oxidants, gums,coating agents, colouring agents, flavouring agents, plasticizers,preservatives, suspending agents, emulsifying agents and spheronizationagents, or any combination thereof.
 8. The method as claimed in claim 6,wherein said subject is an animal, including a human.
 9. The method asclaimed in claim 6, wherein said composition is administered to animalsat a dosage ranging from about 1 mg/kg to about 360 mg/kg.
 10. Themethod as claimed in claim 6, wherein said composition is administeredto humans at a dosage ranging from about 1 mg/kg to about 60 mg/kg.